The pathway for the biosynthesis of saturated fatty acids is very similar in prokaryotes and eukaryotes. However, the organization of the biosynthetic apparatus is very different. Vertebrates possess a type I fatty acid synthase (herein “FAS”) in which all of the enzymatic activities are encoded on one multifunctional polypeptide, the mature protein being a homodimer. The acyl carrier protein (herein “ACP”) is an integral part of the complex. In contrast, in most bacterial and plant FASs (type II) each of the reactions are catalyzed by distinct monofunctional enzymes and the ACP is a discrete protein. Mycobacteria are unique in that they possess both type I and II FASs. There appears to be considerable potential for selective inhibition of the bacterial systems by broad-spectrum antibacterial agents (Rock, C. & Cronan, J. 1996, Biochimica et Biophysica Acta 1302, 1-16; Jackowski, S. 1992. In Emerging Targets in Antibacterial and Antifungal Chemotherapy, Ed. J. Sutcliffe & N. Georgopapadakou, Chapman & Hall, New York; Jackowski, S. et al. (1989), J. Biol. Chem. 264, 7624-7629.)
In the biosynthetic cycle, malonyl-ACP is synthesized from ACP and malonyl-CoA by FabD, malonyl CoA:ACP transacylase. Then, malonyl-ACP is condensed with acetyl-CoA by FabH, acetoacetyl-ACP synthase III. The next step in the elongation cycle is ketoester reduction by β-ketoacyl-ACP reductase (herein “FabG”). Subsequent dehydration by β-hydroxyacyl-ACP dehydrase (herein either “FabA” or “FabZ,” which are distinct enzymes) leads to trans-2-enoyl-ACP which is in turn converted to acyl-ACP by enoyl-ACP reductase (herein “FabI”). In subsequent rounds malonyl-ACP is condensed with the growing-chain acyl-ACP (herein “FabB” or “FabF,” synthases I and IL respectively). Note that gram negative bacteria such as E. coli and Haemophilus influenzae have FabB and FabF enzymes, while at least certain gram positive bacteria, such as staphylococci and streptococci, have only one corresponding enzyme which is most homologous to FabF but functions as FabB. The further rounds of this cycle, adding two carbon atoms per cycle, eventually lead to palmitoyl-ACP whereupon the cycle is stopped largely due to feedback inhibition of FabH and I by palmitoyl-ACP (Heath, et al, (1996), J. Biol. Chem. 271, 1833-1836). The sequence of the E. coli apo-ACP has been described by Rawlings and Cronan (Rawlings, M. and Cronan, J. E., Jr. [1992] J. Biol. Chem. 267, 5751-5754). Moreover, the structures of the acyl-ACP's have been summarized in a review article: Prescott, D. J. and Vagelos, P. R. (1972) Adv. Enzymol. 36, 269-311. Briefly, all of the acyl-ACP's are variants of holo-ACP in which the phosphopantetheinyl group is conjugated with various acyl groups as defined elsewhere herein. The cycle is illustrated in FIG. 1.
Cerulenin and thiolactomycin are potent inhibitors of bacterial fatty acid biosynthesis. Extensive work with these inhibitors has proved that this biosynthetic pathway is essential for bacterial viability. No marketed antibiotics are targeted against fatty acid biosynthesis, therefore it is unlikely that novel antibiotics would be rendered inactive by known antibiotic resistance mechanisms. There is an unmet need for developing new classes of antibiotic compounds, such as those that target bacterial FAS.
Moreover, while acyl-ACP's are known, there is no method for efficiently producing such compounds. In view of their many uses, such as in assays of antibiotic screening using the FAS pathway, there is an unmet need in the art for such methods.